PMC:4067558 / 13234-37045 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4067558","sourcedb":"PMC","sourceid":"4067558","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4067558","text":"Results\n\nExcess Genome-wide Burden of Rare and De Novo Genic CNVs\nTo explore the contribution of CNV to ASD, we expanded our previous study (stage 1)6 with an additional 1,604 families (stage 2), bringing the total to 9,050 individuals from 2,845 ASD-affected families. We used an analytical pipeline of Illumina 1M arrays6,30 to detect rare CNV in families and applied a series of QC filters, including validation of all de novo events by at least one method (Tables S1A–S1C). In total, 1,359 stage 2 families passed QC, and 2,446 families were used in the combined analyses of both stages (Tables S2A and S2B). Of these, 2,147 families were European, and 299 were of other ancestries.21 We used the same pipeline to analyze 2,640 control individuals of European ancestry26,27,29 who were genotyped with the same array platforms. Ancestry was inferred by analysis of SNP genotype data (Table S1B). The rate, size, and number of genes affected by rare (\u003c1% frequency) CNVs were assessed. Consistent with our previous data, we observed that compared to control subjects, affected subjects had an increased burden in the number of genes affected by rare CNVs (1.41-fold increase, empirical p = 1 × 10−5; Table 1). This enrichment was apparent for both deletions and duplications and remained after we controlled for potential case-control differences (Table 1). Similar findings were obtained when each stage was considered separately (Tables S3A–S3C).\nArray- and exome-based studies have revealed a substantial contribution of de novo variation to ASD risk,19 prompting us to assess this further. After screening 2,096 trios (of all ancestries), we found 102 rare de novo CNVs in 99 affected subjects (three of whom had two events; Table S4). Overall, 4.7% of trios had at least one de novo CNV, whereas control subjects had a frequency of 1%–2%.4,31,38 The average length of de novo events in our affected subjects (1.17 Mb) was larger than that of de novo CNVs in unaffected siblings from the Simons Simplex Collection (0.67 Mb, p = 0.01)4 and in control trios (0.55 Mb, p = 0.01).31 The average size of de novo CNVs was also larger than the size of all rare CNVs in our affected (188 kb) and control (159 kb) subjects. De novo CNVs affected 3.8-fold more genes in affected subjects than in control subjects4,31 (2.6-fold for deletions and 6.1-fold for duplications). Even after controlling for the difference in CNV size by proportionally scaling the number of intersected genes in each group, we observed a 1.77-fold difference (1.2-fold for deletions and 2.8-fold for duplications, p = 0.02). Furthermore, de novo CNVs in simplex families intersected 4.0-fold more genes than did CNVs in controls4,31 (1.8-fold after size correction, p = 0.01). There were no significant differences between subjects from simplex families and those from multiplex families in the frequency (5% and 4.2%, respectively) or gene content (n = 18.7 and 18.8, respectively) of de novo CNVs. Similarly, no significant difference was found between males and females in the size (1.17 and 1.2 Mb, respectively) or gene content (n = 18 and 17.3, respectively) of de novo CNVs. For 85 of 102 de novo events, it was possible to determine the parent of origin, and roughly equal numbers of events originated on the paternal allele (n = 45) and the maternal allele (n = 40) (Tables S5A–S5H). Taken together, our data indicate that there is an increased burden of de novo events in ASD-affected subjects. The clinical relevance of de novo CNVs in ASD is confirmed by the fact that among 102 such events identified, half (n = 46) are considered etiologically relevant, including 40 loci known to be involved in ASD and ID (see below).\nWe replicated previous observations, such as a de novo deletion intersecting PTCHD1AS in a male (adding to the evidence that both PTCHD1 and PTCHD1AS contribute to ASD risk14) and de novo events involving the miRNA miR137 (MIM 614304) in 1p21.2–p21.3 in two subjects. Microdeletions of miR137 have been reported in ASD,39 ID,40 and schizophrenia.41 Examples of ASD candidate genes identified by small de novo CNVs include SETD5, DTNA (MIM 601239), and LSAMP (MIM 603241) (Supplemental Data section “Highlighted Genes,” Figures S9, S10, and S14).\n\nCNV Burden in Autosomal-Dominant or X-Linked Genes and Loci Implicated in ASD and/or ID\nAt least 124 genes and 55 genomic loci have been implicated in ASD to date (Tables S6A and S6B; updated from Betancur18), all of which have also been implicated in ID. In addition, we compiled a list of genes and loci that have been implicated in ID, but not yet in ASD (Tables S6C–S6D). When we analyzed samples of inferred European ancestry, we found that 4% (87/2,147) of ASD-affected subjects had CNVs overlapping autosomal-dominant or X-linked genes and loci implicated in ASD and/or ID; this percentage was significantly higher than that in controls (OR = 4.09, 95% confidence interval [CI] = 2.64–6.32, p = 5.7 × 10−12; Figure 1A). We further classified these events into pathogenic, uncertain, or benign according to the American College of Medical Genetics guidelines.32 Pathogenic (or clinically significant) CNVs were identified in 2.8% (60/2,147) of affected subjects (OR = 12.62, 95% CI = 5.44–29.27, p = 2.74 × 10−15), and pathogenic deletions showed a striking estimated OR of 23.13 (95% CI = 5.57–96.08, p = 2.6 × 10−11; Figure 1B). Furthermore, the enrichment of pathogenic CNVs overlapping genes involved in ASD and/or ID was independently observed when the data were broken down by stages: 2.6% (25/979) of affected subjects in stage 1 (OR = 7.61, p = 1.22 × 10−5) carried pathogenic CNVs, whereas 3.0% (35/1,168) in stage 2 (OR = 6.47, p = 2.89 × 10−7) carried pathogenic CNVs. Some of these CNVs (e.g., NRXN1 deletion, 1q21 duplications [MIM 612475], and 16p11.2 duplications) were seen in a small fraction of control subjects, consistent with their variable expressivity and/or incomplete penetrance. Among the affected subjects with pathogenic CNVs, 63% (38/60) carried de novo events (Figure 1C), including two subjects with two pathogenic events each.\nWhen we further considered affected subjects of all ancestries (n = 2,446) and included chromosome abnormalities (\u003e7.5 Mb), select large rare de novo events, and select experimentally validated smaller CNVs (\u003c30 kb), we identified pathogenic CNVs in ∼3.3% of individuals with unexplained ASD (84 pathogenic events in 82/2,446 subjects; Figures 2A and S1A–S1C; Tables S7A and S7B). This most likely represents an underestimate of the true etiologic yield, given that many of the subjects were assessed with clinical diagnostic methods and excluded if positive; similarly, those individuals with known congenital malformations or dysmorphic features were not enrolled. Interestingly, 83% (64/77 [5 without information]) of carriers of pathogenic CNVs were nonsyndromic (i.e., ASD without reported accompanying physical or neurological abnormalities), and 57% (44/77 [5 without information]) had no ID (Figure 2B). The fraction of subjects with ID among carriers of pathogenic CNVs (42%) was not significantly different from the fraction of ID among all affected subjects (46%).\nInheritance data showed that 64% (54/84) of pathogenic CNVs were de novo events (59% were deletions, and 41% were duplications) and that the remaining (36%) were inherited, including seven X-linked CNVs maternally transmitted to males and 23 (13 maternal and 10 paternal [27%]) on autosomes (Figure 2C). Pathogenic deletions tended to be smaller than duplications (Figure 2D). As expected, pathogenic de novo events were on average significantly larger than inherited ones (3.14 Mb—excluding three affected subjects with whole-chromosome aneuploidy—versus 1.44 Mb, respectively). We also observed that the proportion of females was significantly increased among carriers of highly penetrant pathogenic CNVs (male-to-female ratio of 2:1 versus 6:1 among all affected subjects; two-tailed Fisher’s exact test p = 0.017; Figure 2E). In contrast, the male-to-female ratio among individuals with CNVs associated with variable expressivity was 6:1.\nPathogenic CNVs included well-characterized highly penetrant disorders associated with de novo CNVs, such as Phelan-McDermid syndrome (MIM 606232, 22q13.3 deletion including SHANK3 [MIM 606230]), Smith-Magenis syndrome (MIM 182290, 17p11.2 deletion including RAI1 [MIM 607642]), Kleefstra syndrome (MIM 610253, 9q34.3 deletion including EHMT1 [MIM 607001]), Williams syndrome (MIM 194050, 7q11.23 deletion), and large chromosomal abnormalities (Figure 2A; Table S7B). Recurrent deleterious CNVs mediated by segmental duplications affecting 12 distinct regions were identified in 44 individuals. For example, two unrelated males were found to harbor Xq28 duplications (MIM 300815), one de novo and one maternal, corresponding to a ∼0.3 Mb segmental-duplication-mediated gain (153.2–153.5 Mb), which was previously reported in X-linked ID.42 GDI1 (MIM 300104), mutations of which are linked to ID, is the most likely gene involved (Figure S8). Thus, our findings implicate abnormal GDI1 dosage in ASD. Interestingly, one AGP proband with the duplication had autism and a normal IQ, whereas the second had a borderline IQ (72) (see Table S8 for phenotype information of all affected subjects with pathogenic CNVs). Some other findings include a 1.7 Mb de novo deletion encompassing ARID1B (MIM 614556), recently implicated in ID and Coffin-Siris syndrome (MIM 135900), and a small maternally inherited intragenic deletion of HDAC4 (MIM 605314), involved in brachydactyly-mental-retardation syndrome (MIM 600430; Figure S7). Although many 2q37 deletions have been described in ASD, the deletion found in our proband directly implicates HDAC4 haploinsufficiency in autism.\nIn Table S9, we analyzed data across three ASD cohorts, including a total of 5,106 nonoverlapping affected subjects and 3,512 control subjects from the AGP, the Simons Simplex Collection, and the Autism Genetic Resource Exchange (AGRE), for 17 loci and genes commonly reported as implicated in ASD. The most frequent deletions involved 16p11.2 and NRXN1, accounting for 0.31% and 0.32% of affected subjects, respectively. Typical 15q11–q13 duplications of the imprinted PWS-AS critical region were found in 0.25% (13/5,106) of affected subjects, reaffirming this region’s importance in ASD. The majority of these duplications were of maternal origin, but two were paternally derived (one without information; Table S9). Although paternally derived duplications appear to have incomplete penetrance in comparison to maternal ones, there have been several cases reported in subjects with ASD.43\n\nFMRP Targets, PSD Genes, and Other Neuronal Genes Are Implicated in ASD\nWe expanded our analysis to lists of genes important for neurological function, such as highly-brain-expressed genes, PSD genes,33 genes implicated in neurological diseases,44,45 genes with a high pHI,35 and FMRP targets,34 the latter of which have been reported to be enriched in de novo LoF SNVs.7 Our analysis focused on exonic events, and deletions and duplications were analyzed separately (Figures 3A and 3B).\nFMRP targets (n = 842) and PSD genes (n = 1,453) carried a significant excess of both deletions and duplications in affected subjects (Figures 3A and 3B). Five percent (73/1,486) of affected subjects with exonic CNVs, including 52 subjects with genes not previously implicated in ASD and/or ID, carried deletions overlapping one or more FMRP targets, yielding 43 ASD candidate genes (Figure 3A; Table S10). Given that the lists of FMRP targets and PSD genes shared 279 genes, we performed conditional analyses showing that the excess of affected subjects carrying deletions overlapping PSD genes was independent of the signal in FMRP targets (OR = 2.62, 95% CI = 1.62–4.32, p = 2.24 × 10−5) and represented 4% of subjects with exonic events (59/1,486) or 3% after exclusion of pathogenic events (p = 0.007). Notably, females were overrepresented among affected subjects carrying exonic deletions overlapping FMRP targets (17 females in 73 affected subjects, 1.98-fold more than males, p = 0.022, 95% CI = 1.06–3.52).\nBrain-expressed genes showed significant excess in affected versus control subjects for deletions only (OR = 1.89, 95% CI = 1.51–2.37, Fisher’s exact test p = 2.6 × 10−8; Figures 3A and 3B). Similarly, deletions (and not duplications) overlapping genes implicated in dominant neurological diseases and orthologous genes associated with abnormal phenotypes in heterozygous knockout mice conferred significant increase in ASD risk (OR = 2.94, 95% CI = 1.76–4.93, p = 2.5 × 10−5). Many of the genes implicated in dominant diseases have been related to loss of function or haploinsufficiency, previously suggested to be more frequent and penetrant when deletions rather than duplications are involved.46 Accordingly, we detected an excess of affected subjects carrying deletions overlapping genes with a high pHI (\u003e0.35) (OR = 1.41, 95% CI = 1.13–1.76, p = 0.002).\n\nIncreased Multigene Burden in ASD-Affected Subjects\nWe tested whether multiple genes within a CNV or across unlinked genetic lesions in the same individual might act in concert to increase risk of ASD. In logit modeling, the number of genes overlapped by CNVs, the average brain-expression value for those genes, and the deletion or duplication status were used as predictors with the case-control status as the outcome (Figures 3C and 3D).\nWe found that ASD risk increased (as measured by the predicted OR) as the numbers of deleted brain-expressed genes increased (generalized linear model chi-square goodness of fit, p = 3.2 × 10−5 and p = 4.7 × 10−7, respectively; Figures 3C and 3D). These results were consistent across the various models tested (Tables S11A–S11E). There was a decrease in signal after removal of affected subjects with at least one de novo event, suggesting that most of the risk can be attributed to de novo CNVs. Notably, the signal further decreased 2-fold when the remaining pathogenic CNVs were removed, confirming that pathogenic inherited CNVs alone also carry risk. Moreover, we found that gene density contributed significantly to increased risk only when de novo CNVs and inherited pathogenic CNVs were considered, whereas a higher-than-average level of gene expression in deletions (but not duplications) was a contributor irrespective of CNV status (i.e., even after removal of both de novo and inherited pathogenic CNVs) (Tables S11A–S11E). Thus, it is likely that de novo and pathogenic CNVs contribute to risk by altering the expression of more than one gene, suggesting that genetic interactions between these genes can underlie ASD risk.\n\nNetwork Analysis Links Exonic Deletions to Neurodevelopmental Processes\nWe performed a gene-set enrichment analysis6 on our expanded sample set after refining our criteria to consider only exonic events (see Supplemental Data for details) and found only deletions to be significantly enriched in gene sets in affected versus control subjects (Figure 4A). We found 86 significantly enriched gene sets, including MAPK signaling components and neuronal synaptic functions and processes, in 42.5% (335/789) of affected subjects with exonic deletions (Figure 4A; Tables S12A–S12D). Enrichment of synaptic functioning has also been reported among inherited events in the AGRE families47 and among de novo events in the Simons Simplex Collection.36 Enriched sets delineate candidate genes disrupted by deletions not found in control subjects; these genes notably include those in the KEGG glutamatergic pathway (e.g., GRIK2 [MIM 138244], GRM5 [MIM 604102], SHANK1 [MIM 604999], SHANK2 [MIM 603290], and SHANK3 [MIM 606230]; Figure S2A and Tables S13A–S13D), the KEGG cholinergic pathway (e.g., KCNJ12 [MIM 602323], CHAT [MIM 118490], and SLC18A3 [MIM 600336], the latter two of which are within a recurrent 10q11.21–q11.23 deletion, recently reported in individuals with ID and ASD;48 Figure S2B and Tables S13A–S13D), or in both pathways (e.g., GNG13 [MIM 607298], PRKACB [MIM 176892], PLCB1 [MIM 607120], CAMK2G [MIM 602123 ], and PPP3CB [MIM 114106]). When analyzing human homologs of mouse genes, we also found enrichment of phenotypes mostly related to the brain, including abnormal telencephalon morphology, neuron morphology, behavior, and nervous system physiology (Table S12D).\n\nGenes within De Novo CNVs Cluster in a Gene Network\nDe novo deletions (52 in European affected subjects) were found to be significantly enriched within each gene set or pathway cluster (for 85 of the 86 gene sets or pathways), as well as across clusters (chi-square test p = 9.8 × 10−9) (Table S12B), prompting us to search for enriched biological functions within de novo events separately. Taking into account our observations of a significant multigene burden in ASD subjects (Figures 3C and 3D), we analyzed de novo events by using NETBAG36 to identify up to two ASD candidate genes per CNV among 102 de novo events in 99 subjects. NETBAG identifies networks of genes under the premise that if genomic regions are perturbed by genetic variants associated with the same phenotype, they will contain genes forming connected clusters. The NETBAG analysis resulted in a network of 113 genes (global cluster p value = 0.02; Figure 4B). Ten genes have been previously implicated in autosomal-dominant or X-linked forms of ASD and ID (UBE3A [MIM 601623], NRXN1, SHANK2, EHMT1, SYNGAP1 [MIM 603384], and SMARCA2 [MIM 600014]) or ID only (ZEB2 [MIM 605802], FLNA [MIM 300017], SKI [MIM 164780], and IKBKG [MIM 300248]). On the basis of cumulative evidence from various sources, an additional 68% (67/98) are likely to affect ASD risk (Tables S14A–S14E); 27/67 of these are either FMRP targets or PSD genes. Compared to all other genes within de novo CNVs (or deletion CNVs only), genes in the network exhibited a significantly higher pHI (Wilcoxon rank sum p = 7.07 × 10−8), and 55% (59/107 [6 without information]) had a pHI \u003e 0.35. A similar NETBAG analysis of de novo CNVs in control subjects did not yield significant results.36\nWe further characterized the biological processes related to the NETBAG cluster (Tables S14B–S14E; Figures 4B and S3) and found a significant enrichment (false-discovery rate [FDR] \u003c 10%) of genes involved in chromatin and transcription regulation, MAPK signaling, and synaptic signaling and components (Figure 4B). We recapitulated many of the results of our gene-set analysis (Figure 4A), notably for synapse functions and processes, and also identified genes involved in chromatin and transcription regulation. The latter category included a high-risk gene associated with ASD, the chromatin gene CHD2 (MIM 602119), which is affected by a de novo 83 kb deletion in a male with ASD, mild ID, and dysmorphic features including micrognathia and protruding ears. His ASD-affected brother has mild ID, similar dysmorphic features, and epilepsy with onset at age 9 years and carries the same deletion, which removes the first six exons of CHD2. Neither parent carries the deletion, suggesting germline mosaicism (the deletion arose on the paternal chromosome). De novo SNVs in CHD2 have been reported in an ASD subject and in several individuals with a broad spectrum of neurodevelopmental disorders, including ID and epileptic encephalopathy8,49,50 (Figure S6). Two other genes in the chromodomain family have been linked to neurodevelopmental disorders: CHD7 (MIM 608892) in CHARGE syndrome (MIM 214800) and CHD8 (MIM 610528) in ASD. Another example is TRIP12 (MIM 604506), encoding an E3 ubiquitin ligase that can regulate chromatin function to maintain genome integrity (Figure S12). The chromatin and transcription module showed a predominance of genes with a prenatally biased expression profile (Figures 4B and S4).\n\nDe Novo CNVs and LoF SNVs Converge on Functional Gene Networks\nWe expanded our analysis to genes altered by both our de novo CNVs (Figure 5A) and de novo LoF SNVs compiled from four exome sequencing studies in ASD.7–10 Eleven genes affected by de novo CNVs (NRXN1, SHANK2, ARID1B, RIMS1 [MIM 606629], TRIP12, SMARCC2 [MIM 601734], DLL1 [MIM 606582], TM4SF19, MLL3 [MIM 606833], PHF2 [MIM 604351], and CSTF2T [MIM 611968]) were found to be altered by de novo LoF SNVs among autism cohorts, and three of them (NRXN1, SHANK2, and RIMS1) were selected by NETBAG (Figure 4B). Of the 11 genes, only TM4SF19 (3q29) belongs to a known locus associated with a recurrent genomic disorder. Despite the limited overlap observed among genes altered by de novo CNVs and LoF SNVs, there is reinforcing evidence of the role of NRXN1 and SHANK2 in ASD (Figure 5A). In addition, RIMS1, altered by both a de novo duplication and a LoF SNV (Figure 5A) and encoding a brain-specific synaptic Rab3a-binding protein, emerges as an ASD candidate gene. RIMS1 has a regulatory role in synaptic-vesicle exocytosis modulating synaptic transmission and plasticity.51 Three other genes affected by de novo CNVs (also picked by NETBAG)—CHD2, SYNGAP1, and SYNCRIP—are also affected by LoF de novo SNVs in ID,50,52 and two (SYNGAP1 and DPYD [MIM 612779]) are altered in schizophrenia.53 CHD2 (discussed above) and SYNGAP1 (encoding a Ras/Rap GTP-activating protein) are both involved in autosomal-dominant ID, ASD, and epilepsy.54\nUnder the assumption that different genes harboring suspected causative mutations for the same disorder often physically interact, we sought to evaluate protein-protein interactions (PPIs) encoded by genes known to be implicated in ASD and genes affected by rare CNVs or SNVs (data drawn from our de novo CNVs and published de novo ASD LoF SNVs; Figures 5A, 5B, and S5). The union set of 336 unique genes (Table S15) analyzed by DAPPLE37 resulted in a network of direct PPIs encoded by 151 genes (Figure 5A) from each of the three main lists: 54/92 (58.7%) genes involved in ASD, 64/113 (56.6%) de novo CNV genes, and 41/122 (33.6%) de novo LoF SNV genes. The number of direct PPIs was 1.5-fold higher than expected (p \u003c 0.001, Figure 5B), suggesting that many of the de novo CNV or SNV genes and ASD-implicated genes function cooperatively. Overrepresentation analysis identified convergent functional themes related to neuronal development and axon guidance, signaling pathways, and chromatin and transcription regulation. Overall, these findings are consistent among the three different types of analyses shown in Figures 4A, 4B, and 5.\nAlthough 54 genes were previously implicated in ASD, the DAPPLE analysis singled out an additional 97 CNV or SNV high-confidence candidate genes (Figure 5B; Table S16). We found that compared to the 54 ASD-implicated genes, the newly selected 97 CNV or SNV genes had a comparably high pHI (median pHI = 0.58, Figure 6A). This is consistent with the observation that ASD subjects have more deletions with haploinsufficient genes than do controls (Figure 6B). Furthermore, similar to genes with known disease-causing mutations (Figure 6C), those genes have high functional-indispensability scores and a comparable high degree of centrality (i.e., high number of direct neighbors) and number of networks in which they are involved (Figures 6D and 6E). Compared to the genome average, they are also among the top 75% of more-conserved genes (on the basis of Genomic Evolutionary Rate Profiling scores and PhyloP) and are highly expressed in the brain. Interestingly, 39 of the 97 genes are either FMRP-related or PSD genes (of the initial 151 genes identified by DAPPLE, 51 are FMRP interactors and 24 are PSD genes). 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